Cell and Tissue Journal

Abstract Intoduction: Cancer as one of the most common genetic diseases is the leading cause of death worldwide. Cancer cells undergo various genetic and phenotypic changes to spread and survive. In the early stages, these changes lead to the development of tumor, while at the advanced stages they can provide a suitable pre-metastatic microenvironment in which various uncontrolled events occur including cell proliferation, traversing through the extracellular matrix, and crossing barriers to enter the bloodstream. Extracellular changes in this microenvironment can induce intracellular changes in primary cancer cells that assist in the sustainability and propagation of these cells. Complicated interactions between the external and internal factors result in the establishment of various regulatory networks between different types of carcinogenesis promoting factors. Identification of these modifications plays a critical role in understanding the mechanisms of disease progression, prognosis and management. Text: Various mutations and differential gene expression trigger metastasis of cancer cells by epithelial to mesenchymal transition (EMT) mechanism, among which the role of chromatin structural changes, intracellular signal transduction pathways, regulation of cell cycle and microRNAs, and genomic instability has been reported. The alterations in gene expression patterns of mentioned pathways lead to potential regulatory complications that faced the management of disease progression and response to therapies with problems. Cancer cells provide their requirements by neutralizing biological barriers, modifying the regulation of inhibiting processes of cancer progression, establishing de novo endogenous mechanisms and providing specialized molecular and structural markers, and various combinations of these methods have been demonstrated in different types of cancer. Furthermore, EMT and cancer stem cells (CSCs) have a mutual relationship in which the presence of one assists the occurrence of the other. Altogether,  cancer cells take the advantage of  multiple approaches including  upregulation of main transcription factors such as snail, slug, Foxc2, Twist and ZEB1/2, benefiting the mechanisms of telomere length protection, production of CD133,CD44 and BMI1 biomarkers, mutation in P53 coding gene, Failure in acquiring aging phenotype, mutation in amino acid residue S115 of SIM2S gene and increased genomic instability, enhanced activity of signaling pathways such as NF-κB, TGF-β, Wnt, Notch and Hh, mitotic rounding process, facilitated cell division, epigenetic changes such as acetylation and methylation of histones and dysregulation of miRNAs. Conclusion: EMT plays a crucial role in cancer progression, crossing the cells through the biological and body barriers, and metastasis that are usually associated with poor prognosis of cancer patients. Molecular and cellular changes in the main pathways of cells^, development are considered as the promoting factors of EMT and resulted from the differential expression of genes in EMT compared to the normal phenotype of cells. Advancement in the exploration of these changes and their role in the progression of cancer can remarkably affect the early diagnosis, treatment and management of disease. Aim: In this review, various molecular and cellular mechanisms involved in EMT progression and cancer have been investigated, including signal transduction pathways, structural changes of chromatin and telomeres, up/down-regulation of small non-coding RNAs such as miRNAs, cell cycle regulation and genomic instability.

Abstract Aim: Previous studies have shown that Autophagy (lysosome-dependent self-degradation) is upregulated in white adipose tissue of obese subjects. Autophagy-related proteins i.e ATG5 and ATG7 play an essential role in the early stage of the autophagic process. On the other hand, it was found that exercise training modified the bad regulation and maladaptation of white adipose tissue by many molecular mechanisms. Therefore, a question remains to be elucidated whether exercise training can modulate the upregulation of Autophagy induced by a high-fat diet and Autophagy seen in obese subjects. Thus, the present study aimed to survey the effect of regular exercise training on gene expressions of ATG5 and ATG7 in white adipose tissue of mice fed a high-fat diet.
Material and Methods: Twenty-one C57BL/6 male mice (age of four weeks; Approximate body weight of 12 grams) were purchased from the experimental and comparative studies center of Iran University of Medical Sciences. The mice were randomly assigned to three groups: Control (C, n=7), 2) High-fat diet (HFD, n=7), and  High-fat diet with exercise training (HFD-ET, n=7). The mice of the HFD group were fed a high-fat diet (42% kcal of fat) for 12 weeks. The mice of the HFD-ET group were submitted to continuous running on a treadmill for six weeks along with feeding HFD. After the experiment, mice were sacrificed, and visceral adipose tissue pads (epididymal fat) were surgically collected. The Real-Time–PCR methods were used to measure the mRNA expression of ATG5 and ATG7. Data of research were statically analyzed by One-way Analysis of Variance (ANOVA) followed by Tukey's post hoc test.
Results. Data showed that the mRNA expression of ATG5 and ATG7 were higher over two-fold in the HFD group as compared to the control group (p<0.05). Further, the mRNA expression of these genes was higher in the HFD-ET group compared to the control group (p<0.01). Interestingly, the mRNA expression of ATG7 was 1.5 fold higher in the HFD-ET group compared to the HFD group (p<0.05). However, the mRNA expression of ATG5 was not significantly changed in the HFD-ET group as compared to the HFD group (p>0.05).
Conclusion. These results indicate that long-term feeding high-fat diet causes upregulating of the gene expression of key factors involved in the early stage of the autophagy process (i.e ATG5 and ATG7). Regular exercise training could augment the HFD-induced upregulation of ATG7 gene expression. However, it could not change the HFD-induced upregulation of ATG5 gene expression. Based on the results could be speculated that exercise training accompanied by a high-fat diet may more stimulate the autophagy mechanism in white adipose tissue, probably resulting in a positive adaptation in white adipose tissue development. 

Abstract Aim: In recent years using of Bone marrow mesenchymal stem cells (BMSC) in regenerative medicine, tissue engineering and gene therapy is highly regarded. Convenient access, ability to expand and MSC differentiation capacity along with the ability of adhesion to plastic surfaces and in-vitro growth and development are considered as the characteristic feature of these cells. Bone marrow mesenchymal stem cells possess the ability to differentiate into mesodermal lineage, among other adult cells, can be used in tissue engineering and are good candidates for transplantation. Lovastatin as a lowering cholesterol agent and reducing inflammation, as well as antioxidant and, in particular, neuroprotective effects can be effective in the treatment of neurogenic diseases. The aim of this study was to evaluate the effect of lovastatin on survival, proliferation and expression of GDNF and oct4 genes of Bone marrow mesenchymal stem cells. Lovastatin is presumed to exert their neuroprotective effects by inducing neurotrophic factor gene expression and cell proliferation. Material and methods: In this experimental study, we used 4-6 week adult Wistar rats. The BMSCs were isolated from rat femurs and tibias and cultured in α-MEM. The cell pellet was resuspended in α-MEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and cultured in 25-cm2 culture flasks at a density of 2 × 104 cells and incubated at 37°C and 5% CO2.  For lovastatin treatment, we exposed MSCs to 1 μM, 5 μm, 10 μm and 15 μm of lovastatin for 24 h. The survival rate of cells was measured by MTT assay. The growth rate and proliferation of cells at 24 hours after culture were assessed by staining with DAPI. The expression of Oct4 and GDNF factors was also evaluated by RT-PCR.
 Results: MSCs were attached to culture plate and were quickly proliferated. In the culture plate, these cells were usually appeared in three forms: small spherical, fusiform and fibroblast-like and flattened. The results of this study indicate that the proliferation rate at 5, 10 and 15 µM lovastatin showed a significant increase compared to control groups (P<0.5). Gene expression density of gelial derived neurotrophic factors (GDNF) and oct4 genes showed that, there were significant differences between MSCs treatment groups and control group (P<0.5). Cell viability and proliferation rate indicate that experimental groups has a higher proliferation rate than control group. Moreover, results showed an increase in mRNA expression for GDNF and Oct4 compared to the control group (P <0.05).
 Conclusion:Therefore, lovastatin can be used to improve the culture of mesenchymal stem cells, which is used for transplantation and cell therapy. BMSCs may be a useful therapeutic agent for the treatment of neurodegenerative disorders.

Abstract Aim: The three-dimensional (3D) culture of cancer cells is a method that provides the possibility for growth and comprehensive communication of cells in a 3D space, leading to the generation of tumorspheres. In recent years, developing tumor models from cancer cells by using the 3D cell culture method has attracted a lot of attention because it has been introduced as an accurate and reliable strategy for studying cancer stem cells (CSCs) and CSC-based therapeutics. The 3D tumor models in comparison to the monolayer culture (two-dimensional (2D) culture) of cells more resemble in vivo conditions. Because in tumor models, the tumor microenvironment, cell to cell and cell to extracellular matrix interactions and hypoxia condition, which is necessary for the survival of CSCs, are well reproduced. Through the use of several types of cells, including cancer and stromal cells, tumor models have the ability to develop and reflect the complexity of the tissue of interest, which in such a case, are even more accurate models in reflecting the biochemical and physical conditions of the body. Therefore, in the present study, the 3D model of breast cancer has been constructed with the aim of investigating the relationship between the cell behavior and the cell culture conditions (2D and 3D), and a comparative study of the growth and drug response of the two human breast cancer cell lines; MCF-7, and MDA-MB-231. Material and Methods: The two breast cancer cell lines, MCF-7, and MDA-MB-231, were cultured in 2D and 3D (in two modes; on top and embedded) on the Matrigel-based scaffold. The molecular phenotype of cells based on surface markers was examined by flow cytometry. Mammosphere growth was followed in 12 days and their growth kinetics was determined. To evaluate the drug response of cells, two anticancer drugs; actinomycin D and paclitaxel were applied. Primarily, the IC50 values of the two drugs were evaluated, then the generated mammospheres were treated at the indicated dose of the drugs, and their effect on the growth of the mammospheres was followed.
Results: The MCF-7 and MDA-MB-231 cell lines cultured in 2 and 3D, showed a significant difference in their molecular phenotypes. So, it seems that the expression of CD44 has significantly decreased.  On the other hand, the growth rate of cells in two different modes of 3D culture; on to and embedded, is different. Likely, the drug response evaluation shows a significant difference in 2D and 3D culture, so that the inhibitory effect of paclitaxel compared to actinomycin D has decreased in 3D culture. In addition, the results show that MCF-7 and MDA-MB231 have different drug responses, which can be affected by their different molecular phenotypes. Conclusion: The results of the study confirm that the molecular phenotype of cancer cells, their growth, and drug response are strongly affected by the type of the understudied cell lines, the cell culture method, and the applied drug. Consequently, conducting cancer studies as accurately as possible requires obtaining a model that is most similar to the corresponding tumor in the body.
 

Abstract Aim: Cell therapy using mesenchymal stem cells (MSCs) can be a promising tool in regenerative medicine. One of the richest sources of mesenchymal stem cells is fetal bone marrow. Silymarin has strong antioxidant and anti-inflammatory activities with a positive effect on the proliferation of some cells as well as anti-osteoporosis properties. This study aimed to show the effect of silymarin on the differentiation of mesenchymal stem cells derived from the bone marrow of sheep embryos into the osteogenic line.
Materials and Methods: Mesenchymal stem cells were isolated from the bone marrow of sheep embryos. MTT test was performed to investigate the cytotoxicity of silymarin on cells at different concentrations for 24 and 72 hours. Then, cells in one of 8 groups 1: negative control; 2: treated with 10 μmol/liter silymarin in the usual environment, 3: treated with 20 μmol/liter silymarin in the usual environment, 4: treated with 100 μmol/liter estradiol in the usual environment, 5: positive control, 6: treatment treated with 10 μmol/liter silymarin in the differentiation medium, 7: treated with 20 μmol/liter silymarin in the differentiation medium, 8: treated with 100 μmol/liter in the differentiation medium, were cultured for 21 days. To determine the osteogenic differentiation of cells, the deposition of hydroxyapatite ions was examined using alizarin staining, and also, the amount of ALP enzyme secretion was also measured in the studied groups. Results: Comparing the average optical absorption of cells at different concentrations between 24 and 72 hours after treatment showed that the average optical absorption of cells at zero concentration of silymarin after 72 hours of treatment decreased in comparison with those treated for 24 hours (P<0.05), but no significant difference was observed in other concentrations (P>0.05). Examining the level of ALP enzyme secretion, 21 days after treatment with silymarin in the studied groups showed that the highest level of enzyme secretion was in group 8 (P≤0.05). The lowest amount of enzyme secretion was observed in group 1 (negative control) and then in group 2 and group 3 respectively (P<0.05). No significant difference was observed between groups 4, 5, and 6 (P>0.05). Based on the alizarin red staining results, calcium ions deposition was observed in all the groups related to the differentiation medium, which increased in groups 8, 7, 6, and 5, respectively. In the groups cultured in the usual environment, there was no calcification in group 1 and the amount of calcification increased in groups 2, 3, and 4, respectively. In total, the amount of calcification in the differentiation environment groups was higher in comparison with the usual environment.
Conclusion: During this study, Silymarin had no toxic effect on the mesenchymal stem cells derived from the bone marrow of sheep embryos in the studied concentrations after 24 and 72 hours of treatment. It increased the differentiation of the cells into the osteogenic lineage in a concentration-dependent manner. Therefore, it seems that with further studies and identification of the molecular pathways of silymarin's effect, it can be used in cell therapy in order to repair bone lesions.

Abstract Aim: In the past decades, many efforts have been made with the aim of searching for new tools to treat cancer. In this regard, the discovery, investigation and application of techniques related to small interfering RNAs (siRNA) has been one of the most significant advances in the field of cancer detection and treatment. Small interfering RNA, sometimes known as short interfering RNA or silencing RNA, is usually 21 bp long and interferes with the expression of specific genes with complementary nucleotide sequences and prevents translation by degrading mRNA after transcription. Many studies have shown that siRNAs affect the regulation of the expression of some genes that play a role in cancers. siRNAs are effective on Snail transcription factors, which play an important role in the invasion and metastasis of cancer cells, and miR-143, which plays an important role in the pathogenesis of cancers. miRNAs together with transcription factors can disrupt the biological pathways involved in carcinogenesis. However, the exact effect of siRNA on the expression of snail1 and miRNA-143 genes in breast cancer cells is not completely clear. Based on this, the present study investigated the effects of siRNA on snail1 and miRNA-143 on breast cancer cells.Material and Methods were purchased from Pasteur Institute of Iran. The cells were cultured in RPMI-1640 medium containing 10% FBS. Snail1 gene kit (Santacruz biotechnology, California, USA) was used to treat cancer cells with specific siRNA. The cells were divided into two groups: control (no treatment) and treated cells (transfected with siRNA). In order to determine the effective time, the cells were exposed to a dose of 60 picomoles of siRNA for 24, 48 and 72 hours. Beta actin gene was used as internal control gene. Morphology of MDA-MB-468 metastatic cells were examined using light microscopy before and after specific gene transfection. Cell proliferation was checked by trypan blue staining. Snail1 and miR-143 gene expression levels were evaluated by qRT-PCR. Data were analyzed using t-test. Results: In this study, in MDA-MB-468 breast cancer cells, the relative level of Snail1 gene expression was significantly decreased in the effective time of 48 hours and when exposed to the effective dose of 60 pmol (P < 0.0001). However, the knockdown of Snail1 gene by specific siRNA in MDA-MB-468 cancer cells when exposed to an effective dose of 60 pmol and an effective time of 48 hours caused an increase in the relative expression level of miR-143 gene compared to the control group (P < 0.0001). Also, the growth rate of MDA-MB-468 cancer cells decreased with Snail1 gene knockdown. Conclusion: The results of this research showed that the transfection of MDA-MB-468 breast cancer cells by specific siRNA can successfully reduce the expression level of Snail1 gene and miR-143 gene. Proliferation and invasion of breast cancer cells.